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Mechanism of Surface-Enhanced Raman Scattering Based on 3D Graphene-TiO2 Nanocomposites and Application to Real-time Monitoring of Telomerase Activity in Differentiation of Stem Cells Tingting Zheng, Enduo Feng, Zhiqiang Wang, Xue-Qing Gong, and Yang Tian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11028 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 6, 2017

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ACS Applied Materials & Interfaces

Mechanism of Surface-Enhanced Raman Scattering Based on 3D Graphene-TiO2 Nanocomposites and Application to Real-time Monitoring of Telomerase Activity in Differentiation of Stem Cells Tingting Zheng,†,§ Enduo Feng, †,§ Zhiqiang Wang, ‡ Xueqing Gong, ‡ Yang Tian†,* †

†Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Dongchuan Road 500, Shanghai 200241, China. E-mail: [email protected] ‡ Key Laboratory for Advanced Materials, Centre or computational Chemistry and Research Institute of Industrial Catalysis, School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, P.R. China.

KEYWORDS: Surface-enhanced Raman scattering, graphene, TiO 2 , telomerase activity, stem cells

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ABSTRACT: With a burst development of new nanomaterials for plasmon-free surface-enhanced Raman scattering (SERS), the understanding of chemical mechanism and further applications have become more and more attractive. Herein, a novel SERS platform was specially designed through electrochemical deposition of graphene onto TiO 2 nanoarrays (EG-TiO 2 ). The developed EGTiO 2 nanocomposites SERS platform possessed remarkable Raman activity using copper phthalocyanine (CuPc) as a probe molecule. XPS measurement revealed that the chemical bond Ti-O-C was formed at the interface between graphene and TiO 2 in EG-TiO 2 nanocomposites. Both experimental and theoretical results demonstrated that the obvious Raman enhancement was attributed to TiO 2 -induced Fermi level shift of graphene, resulting in effective charge transfer between EG-TiO 2 nanocomposites and molecules. Taking advantage of marked Raman response of CuPc molecule on EG-TiO 2 nanocomposites surface as well as specific recognition of CuPc toward multiple telomeric G-quadruplex, EG-TiO 2 nanocomposites was tactfully employed as SERS substrate for selective and ultrasensitive determination of telomerase activity with low detection limit down to 2.07×10-16 IU. Interestingly, self-cleaning characteristic of EG-TiO 2 nanocomposites under visible light irradiation successfully provided a recycling ability for this plasmon-free EG-TiO 2 substrate. The present SERS biosensor with high analytical performance such as high selectivity and sensitivity, has been further explored to determine telomerase activity in stem cells, as well as to count the cell numbers. More importantly, using this useful tool, it was discovered that telomerase activity plays an important role in the proliferation and differentiation from human mesenchymal stem cells (hMSCs) to neural stem cells (NSCs). This work has not only established an approach for gaining fundamental insights into chemical mechanism (CM) of Raman enhancement, but also has opened a new way in investigation of long-term dynamic of stem cell differentiation and clinical drug screening.


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INTRODUCTION Surface-enhanced Raman scattering (SERS) is a widely studied and considered as a promising technique for microanalysis and trace species detection due to its remarkable advantages such as high sensitivity, nondestructive, quick response as well as providing unique information on the species.

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Conventional SERS phenomenon is mainly attributed to electromagnetic field

mechanism (EM) generated at the surface of noble metal such as Au and Ag, upon laser excitation. 4,5

Recently, another kind of Raman enhancement has been attracted more attention. The

mechanism of this kind of Raman enhancement, named chemical mechanism (CM), is attributed to the chemical interaction between substrate and the adsorped molecules. The detailed mechanism of chemical Raman enhancement is actually poorly understood at the present stage.

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The

enhancement factors (EFs) originated for EM and CM depend on electromagnetic frequency resonance and molecule-substrate interaction, respectively. 7 EF values dominated by CM usually ranges from 10 to 100, 8,9 which is smaller than that obtained by the conventional SERS method. However, two dimensional SERS substrates originated from CM, including graphene, black phosphorus, hexagonal boron nitride, and molybdenum disulfide, have flat and smooth surfaces particularly suitable for selectively homogeneous deposition of aromatic molecules. Furthermore, the generated Raman signal is exclusive chemical enhancement, providing quantitative and specific analysis with high reproducibility and stability accessible. The star material among them is graphene, owing to its remarkable advantages, such as atomic uniformity, large delocalized bond, unique electronic structures, and chemical inertness. 10,11 Herein, we developed a 3D material through electrochemical deposition of graphene onto TiO 2 nanoarrays (denoted as EG-TiO 2 nanocomposites), which show a remarkable enhancement of Raman scattering compared with native graphene or TiO 2 nanoarrays. Hybrid graphene and TiO 2


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nanomaterials have received increasing attention due to their enhanced photoconversion efficiency, but it is the first time that EG-TiO 2 nanocomposites were developed for SERS substrate. From XPS data, it was found that Ti-O-C bond was formed on the interface between EG and TiO 2 , confirming strong chemical interaction between electrodeposited graphene and TiO 2 nanoarrays. The theoretical and experimental results demonstrated that the electronic structures and Fermi levels of graphene clearly changed in EG-TiO 2 nanocomposites as compared with those of pure electrodeposited graphene (EG). Thus, remarkable Raman enhancement was observed at EG-TiO 2 nanocomposites due to highly efficient charge transfer from the shifted Fermi level to probe molecules. More interestingly, copper phthalocyanine (CuPc), which has a large Raman crosssection, was designed as the probe molecule. CuPc is a planar and π-conjugated molecule. 12 The exact electronic coupling of CuPc molecule to the “aromatic” EG-TiO 2 nanocomposites resulted in high molecular recognition ability. 13 Meanwhile, CuPc is a high affinity anionic ligand of Gquadruplex, 14 a four-stranded DNA structure with large planar aromatic rings (G-quartet) that forms from human telomere DNA consist of short tandem repeats of TTAGGG (in vertebrates). 15 On the other hand, telomere length homeostasis is maintained by telomerase, a ribonucleoprotein that synthesizes telomere repeats onto telomere ends to compensate the natural shortening of the telomeres.

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The higher telomerase elongation efficiency leads to the greater number of G-

quadruplexes. Therefore, intracellular telomerase activity can be tracked by Raman signal of CuPc attached to the G-quadruplexes. Specially, in this work, adopting our EG-TiO 2 SERS platform, the incorporation of CuPc into G-quadruplex telomere structures enabled SERS evaluation of telomerase activity with high sensitivity and selectivity. Significantly, for the first time, this unique SERS biosensor was successfully used in exploring the important role of telomerase activity plays in proliferation and differentiation capacity of stem cells.


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RESULTS AND DISCUSSION Development and Characterization of EG-TiO 2 Nanocomposites. The EG-TiO 2 nanocomposites were first designed and synthesized by electrodeposition of graphene onto TiO 2 nanoarrays. TiO 2 nanoarrays were fabricated using a facile two-step anodization process. 17 A top view scanning electron microscopic (SEM) image of TiO 2 nanoarrays in Figure 1a demonstrates high uniformity of the top nanoring structure in large scale. The magnified SEM image given in the inset of Figure 1a depicts clear periodical nanoring structure with an average diameter of ~200 nm and thickness of ~50 nm. The length of the nanotubes was controlled to ~1.5 μm (cross-section view

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Figure 1. (a) Large-scale and magnification (inset) top view of SEM images of TiO 2 nanoarrays. (b) Cross-section SEM and TEM (inset) images of TiO 2 nanoarrays. (c) TEM image of top photonic nanorings of TiO 2 nanoarrays. (d) High resolution TEM image of TiO 2 nanoarrays. (e) Large-scale and magnification (inset) top view of SEM images of EG-TiO 2 platform. (f) XRD patterns (g) Raman spectra, and (h) FTIR spectra of (I) EG, (II) TiO 2 and (III) EG-TiO 2 nanoarrays.


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maximum penetration depth of the incident light in TiO 2 .18 The top layer of TiO 2 nanoarrays with periodically nanoring structures was clearly observed in transmission electron microscopy (TEM) image of Figure 1c. High-resolution TEM (HRTEM) image (Figure 1d) demonstrates wellresolved lattice fringes with an inter-planar distance of 0.35 nm, which corresponds to (101) plane of anatase TiO 2 . 19, 20 Then, graphene was electrochemically deposited onto the TiO 2 nanoarrays surface.

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A typical cyclic voltammetry (CV) of graphene oxide (GO) electrolysis on TiO 2

nanoarrays was employed to track the electrodeposition process (Figure S1, Supporting Information). The cathodic peak I was attributed to the irreversible electro-chemical reduction of GO, while the cathodic peak II and the anodic peak III were ascribed to the redox pair of oxygencontaining groups on the graphene plane. 22 As shown in Figure 1e, a continuous and transparent graphene layer was well-coated on the surface of TiO 2 nanoarrays. Additionally, full-wavelength Raman spectrum of EG-TiO 2 nanocomposites (Figure S2a) was carried out to make a determination of the number of layers of graphene sheet. The Raman spectrum of EG-TiO 2 nanocomposites clearly showed a strong D band at 1330 cm-1, a slightly weak G band at 1595 cmrelative to the D band and a very weak 2D band at 2710 cm-1, which is the characteristic of single-

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layered oxidative graphene Raman spectra. 23 In other hand, AFM image of EG-TiO 2 (Figure S2b) with a thickness for ~0.6 nm also validated that graphene was deposited on TiO 2 as single layer. The crystalline structures of TiO 2 and EG-TiO 2 nanocomposites were further characterized by powder X-ray diffraction (XRD) spectroscopy (Figure 1f). A series of diffraction peaks were clearly observed at 25.28°, 37.80°, 38.58°, 48.05°, 53.89° and 55.06° in the wide-angle XRD pattern for TiO 2 nanoarrays, corresponding to (101), (004), (112), (200), (105) and (211) planes of anatase phase TiO 2 nanocrystals.

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For EG-TiO 2 nanocomposites, two additional weak

diffraction peaks were obtained at 21.2° and 43.3°, which were resulted from the electrodeposited


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graphene as shown in Figure 1f. 25 Furthermore, as depicted in Figure 1g, both TiO 2 and EG-TiO 2 nanocomposites show characteristic Raman active modes at 397 cm-1, 518 cm-1 and 640 cm-1, corresponding to the existence of anatase phase. 26 However, two distinct peaks, D band at 1330 cm-1 and G band at 1595 cm-1, both appeared at EG and EG-TiO 2 nanocomposites, indicating two typical Raman active vibration modes in graphitic structures.

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In FTIR spectra (Figure 1h),

compared to TiO 2 nanoarrays, EG-TiO 2 nanocomposites show additional infrared bands assigned to aromatic ring frame vibration at 1750 cm-1, N=C=O stretching vibration at 2270 cm-1, and the CH 2 - bending vibration at 2910 cm-1.

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Both Raman and FTIR results reveal that graphene has

been successfully electrodeposited on the surface of TiO 2 nanoarrays. More importantly, X-ray photoelectron spectroscopy (XPS) was performed to elucidate the chemical state of elements in EG-TiO 2 nanocomposites. From XPS survey spectrum shown in Figure. 2a, it is clear that EG-TiO 2 nanocomposites mainly consist of C, O and Ti without trace of contamination. The high-resolution XPS of C, O and Ti in EG, TiO 2 and EG-TiO 2 nanocomposites show distinct profiles (Figure 2b-d). The C 1s spectrum of EG is deconvoluted into C-C (non-oxygenated ring C, 284.6 eV), C-O (285.6 eV), and carbonyl O=C (carboxylic, 287.8 eV) species.

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Binding energies of all these peaks, especially O=C energy, shift to higher

energy levels in EG-TiO 2 nanocomposites (Figure 2b). On the other hand, the Ti core-level XPS spectrum in TiO 2 nanoarrays demonstrates two peaks centered at 458.5 and 464.2 eV (Figure 2c), which are assigned respectively to the Ti 2p1 and Ti 2p3 spin-orbital splitting photoelectrons in the Ti4+ state. 29 For EG-TiO 2 nanocomposites, these two peaks were observed at 459.3 and 465.0 eV, respectively. The splitting energy between two Ti-bands of these both samples was estimated to 5.7 eV, implying the presence of normal state of Ti4+. 30 The difference of 0.8 eV between the Ti peak positions of TiO 2 and EG-TiO 2 nanocomposites was attributed to the interactions between


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Figure 2. (a) XPS survey spectra of EG, TiO 2 and EG-TiO 2 nanocomposites. Core-level XPS spectra of (b) C1s, (c) Ti2p and (d) O1s of EG, TiO 2 and EG-TiO 2 nanocomposites. Ti and oxygen centers of EG. Since oxygen, a highly electronegative element, withdrew the electron density from Ti of EG-TiO 2 nanocomposites. As a result, the binding energy of Ti in EGTiO 2 nanocomposites increased compared with that in TiO 2 . To further support the presence of this binding, O1s core levels were also measured. As shown in Figure 2d, three peaks centered at 529.4, 530.0, 531.1 eV were clearly obtained, which were assigned to the presence of O2-, -OH, and oxygen vacancies (O v ), respectively. 31 The greater ratios of O v (68.15%) indicate more oxygen vacancies in EG, which are always coupling with Ti3+. After binding with TiO 2 , the peak of O v decreased while O2- increased, and binding energy of all these peaks shifted toward higher energy levels. In agreement with previous reports, this peak was assigned to binding energy of O in Ti-O-C bond.

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Thus, it was confirmed that the Ti-O-C

chemical bond was formed onto the interface between EG and TiO 2 in EG-TiO 2 nanocomposites.


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Figure 3. Raman spectra of (a) CC, (b) CV, and (c) CuPc on EG-TiO 2 nanocomposites (red line), EG (blue line), TiO 2 (green line) and on a blank SiO 2 /Si substrate (black line) with the excitation laser wavelength of 633 nm. (d) Statistical data analysis for the comparison between the Raman intensity (1530 cm-1) of CuPc CV and CC. Each data point represents the average value from three replicate SERS spectra (S.D., n= 6). Molecular structures and HOMO/LUMO energy levels of (e) CC, (f) CV, and (g) CuPc using DFT calculation. (h) Simulated models of EG-TiO 2 nanocomposites. Light grey, red, and dark grey, spheres represent Ti, O, and C atoms, respectively. The atoms in the surface layer of TiO 2 (001) have slight movement, and the positive (negative) values represent the atoms that slightly move upwards (push downwards). (i) Proposed mechanism for charge transfer of CC, CV and CuPc in EG and EG-TiO 2 platforms.


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Mechanism of Raman Enhancement in EG-TiO 2 Nanocomposites. The effect of Raman enhancement with CM strongly depends on the matching degree of structure symmetry and energy level between molecules and substrates.

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Figure 3a-c show Raman spectra of three typical

symmetric molecules of D 2h (catechol, CC), C 3 (crystal violet, CV), and D 4h (copper phthalocyanine, CuPc) on EG-TiO 2 nanocomposites (red line), EG (blue line), TiO 2 (green line), and SiO 2 /Si substrates (black line) under laser excitation of 633 nm. Obvious EFs were obtained on EG (9.7 for CC, 25.8 for CV and 20.5 for CuPc) and EG-TiO 2 nanocomposites (14.2 for CC, 24.6 for CV and 48.2 for CuPc) (Table S1) substrates, which were ascribed to symmetry matching between molecules of CC, CV, and CuPc with graphene substrate. On the contrary, no clear EFs were obtained for asymmetric molecules, such as oxalic acid, salicylic acid and dopamine (Figure S3, Supporting Information). Furthermore, the hexagonal lattice surface structure of EG-TiO 2 nanocomposites was similar to parallel-connected rings in the molecular structure of CV and CuPc. The structural similarity between both EG and EG-TiO 2 substrates and the molecules generally reduced the distance between molecules and substrates, thus strengthening the coupling. Such high symmetry and planar structure contribute to the obvious enhancement effects of CuPc and CV molecules on EG and EG-TiO 2 nanocomposites. Focusing on 1530 cm-1 photon mode, the distortion vibration of the 16-membered macrocycle and benzene ring. 34 Figure 3d summarized the Raman intensity of CC, CV and CuPc at 1530cm-1 . It is very interesting that Raman scattering intensity was obviously enhanced on EG-TiO 2 nanocomposites for CuPc (~2.4 fold) and CC (~1.5 fold) molecules while that was slightly decreased for CV molecule, compared with those on EG substrate. Distinctly, the obvious changes should be ascribed to electronic structure between EG-TiO 2 nanocomposites and EG sheet. It is well-known that work function of graphene is larger than that of TiO 2 . Thus, when EG is in contact


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with an n-type semiconductor such as TiO 2 , the charge distribution is readjusted for equilibration of Fermi level between EG and TiO 2 , resulting in Fermi level elevation of EG to form a Schottky barrier (depletion layer) at the junction between these two materials. 35 Compared with the energy difference between Fermi level of EG nanocomposites and LUMO level of CuPc and CC, a smaller difference between Fermi level of EG-TiO 2 nanocomposites and LUMO level of CuPc and CC leads to more effective charge transfer between EG-TiO 2 nanocomposites and molecules, accordingly resulting in stronger Raman scattering. To confirm the experimental results demonstrated above, the following model was employed, as shown in Figure 3g, for estimating Fermi level of EG-TiO 2 system: a 3×2 surface cell was used to construct a three-layer TiO 2 (101) slab, and a 3×7 surface cell for an EG slab. The valence atomic configure rations were Ti: 3d24s2, O: 2s22p4 and C: 2s22p2, respectively. All calculations were performed using DFT methodologies implemented in the Vienna ab-initio simulation package (VASP). As demonstrated in Figure 3h, the Fermi level of EG-TiO 2 system was calculated to be situated at -3.65 eV, which is just between the calculated HOMO/LUMO levels of CuPc and CC. The energy difference between the Fermi level of EG-TiO 2 and LUMO levels of both molecules is smaller than that of EG resulting in an obvious Raman enhancement. Inversely, after introduction of TiO 2 , the Fermi level of EG-TiO 2 nanocomposites shifted higher than LUMO level of CV, leading to a slight decline of Raman intensity. Furthermore, the match between excitation laser energy and HOMO/LUMO gap of molecules is also an important factor for Raman enhancement. HOMO/LUMO gaps of CV and CuPc were estimated to be 1.9 eV and 1.7 eV, respectively, using density functional theory (DFT) (Figure 3de). For CuPc and CV under 633 nm (1.96 eV) laser excitation, the EFs are stronger than under 532 nm (2.33 eV) laser excitation (Table S2), possibly because the 532 nm laser excitation is not in the


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Figure 4. (a) Schematic illustration for working principle of the developed SERS platform based on EG-TiO 2 nanocomposites for determination of telomerase activity. (b) Raman spectra of the developed EG-TiO 2 nanocomposites SERS biosensor obtained with telomerase activity of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 IU L-1(c) Plot of Raman scattering intensity vs telomerase activity. Each data point represents the average value from three replicate SERS spectra (S.D., n= 6). (d) Raman spectra of the EG-TiO 2 nanocomposites SERS biosensor obtained with hMSC concentrations of 10, 102, 103, 104, 105, 106, 107 cells mL-1. (e) Plot of Raman scattering intensity vs logarithm of hMSC concentration. Each data point represents the average value from three replicate SERS spectra (S.D., n= 6). Error bars equal to the standard deviations. (f) SERS imaging of telomerase activity obtained with different hMSC concentrations. resonance window with the HOMO/LUMO energy gap of CuPc and CV. 25 On the contrary, CC has a HOMO/LUMO gap of 4.4 eV (Figure 3f), which is too large to be resonant with 532 or 633


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nm laser excitation. Consequently, no obvious SERS effects were observed for CC under 532 or 633 nm excitation. Imaging and Biosensing of Telomerase Activity in Human Mesenchymal Stem Cells (hMSCs). Besides the high molecular recognition ability of EG-TiO 2 nanocomposites SERS substrate toward CuPc demonstrated above, CuPc is a high affinity anionic ligand of G-quadruplex, which can be produced from human telomere repeats (TTAGGG)n. Therefore, the developed EGTiO 2 nanocomposites was employed as enhanced Raman substrate for both selective and quantitative characterization of telomerase activity as well as for counting of cell numbers using CuPc as a specific recognition element. Figure 4a illustrates the major steps involved in the working principle of the present SERS biosensor for determination of telomerase activity based on EG-TiO 2 nanocomposites substrate. Amino-modified telomerase primers were first immobilized on EG-TiO 2 nanocomposites and extended by telomerase to form telomere repeats (TTAGGG)n. The extension of primer caused by telomerase was verified by gel electrophoresis (Figure S4, Supporting Information). Then, the guanine (G)-rich sequences undergo conformational changes to form a G-quadruplex, 36 which exhibits high selectivity and affinity for CuPc (K d = 42 M in the presence of 100 mM KCl). 37 In this way, intracellular telomerase activity can be tracked through Raman signal of CuPc. The stepwise assembly process of the developed SERS biosensor was monitored by electrochemical impedance spectroscopy (EIS) measurements (Figure S5, Supporting Information), which indicated a successful fabrication of SERS biosensor. After optimizing the experimental conditions (Figure S6, Supporting Information), the activity of pure telomerase was subsequently evaluated by the proposed approach to validate sensitivity and selectivity of this strategy. To exclude unexpected backgrounds which were caused by non-specific adsorption of CuPc, we firstly synthesized a DNA sequence 5’-NH 2 - AAT CCG TCG AGC AGA


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GTT TTA GGG TTA GGG TTA GGG-3’, to simulate the elongated primer sequence by telomerase. As shown in Figure S7a, a very weak Raman signal was obtained from the platform with primer, while a significant enhancement was observed with E-primer. Furthermore, we also employed UV-Vis spectra to explore the non-specific adsorptions of CuPc. CuPc molecules could absorb incident UV-Vis light and showed two obvious adsorption peaks at 620 nm and 690 nm, respectively. Figure S7b showed the representative spectra of EG-TiO 2 . An obvious adsorption peak both at ~620 nm and 690 nm were observed for the E-primer modified EG-TiO 2 . On the contrary, no obvious adsorption peak in the range of 600 nm-700 nm for the primer modified EGTiO 2 . Together with the comparison of Raman spectra, these results indicated that there were few non-specific adsorptions of CuPc on the EG-TiO 2 nanocomposites. In addition, because of the presence of G-quadruplex between CuPc molecules and EG-TiO 2 nanocomposites, the enhancement effect of EG-TiO 2 nanocomposites for CuPc might be affect. To confirm the influence of the DNA nanostructures to Raman signal, we conducted an additional experimental comparison with/without the presence of DNA nanostructures in Figure S7a. There were not obvious differences under these two different conditions, which reflected that DNA nanostructures had negligible influences to the Raman signals. As shown in Figure 4b, Raman scattering intensity increased with the increasing concentration of telomerase. The characteristic peak around 1529 cm-1 was employed to directly probe CuPc attached onto EG-TiO 2 nanocomposites, and thus to determine telomerase activity. The calibration curve displays a linear relationship between Raman scattering intensity (I) and telomerase activity (A) over the range of 2.1x10-15 IU to 2.3x10-11 IU in a single cell (R =0.998, n = 7) (Figure 4c). The linear regression equation was I = 3.905×103 + 763.6 lg (A×1012) (IU)

(1)


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The detection limit was achieved down to be 2.07×10-16 IU at 3σ, which is much lower than other approach for detection of telomerase .38,39 A critical length of telomere repeats is required to ensure proper telomere function and avoid the activation of DNA damage pathways that results in replicative senescence or cell death. As hMSCs have elongated proliferative capacity, telomerase activity level is important to maintain telomere length through many cell divisions. Herein, the proposed EG-TiO 2 nanocomposites platform was further used for hMSC counting as well as evaluation the role telomerase activity played in the proliferation process of hMSCs. Figure 4d displays the Raman signal of biosensor corresponding to the 1st generation hMSCs at different concentrations. The Raman intensity increased monotonically with the logarithm concentration of hMSCs as shown in Figure 4e, and the detection limit was determined to be ~10 cells mL-1 at 3σ. As 100 μL of cell suspension was used for incubation, our approach was used to realize the telomerase activity measurement down to 1 hMSC, which was significantly lower than those electrochemical and fluorescent cytosensing approaches. 40

A topographic image of 100×100 μm2 was also collected from the integrated intensity of 1529

cm-1 peak. As demonstrated in Figure 4f, Raman signals of the developed SERS biosensor for telomerase activity remarkably increased with the increasing concentration of cells. However, negligible Raman signal changes were observed for this SERS biosensor incubated with PBS. The results suggest that CuPc can successfully attach to G-quadruplex extended by telomerase from cell extracts with high selectivity. Recyclability of EG-TiO 2 Nanocomposites SERS Biosensor. As well-known, TiO 2 shows UVvis absorption in the range of 200-350 nm, 41 which was also confirmed in Figure S8 (Supporting Information). However, absorbance of EG-TiO 2 nanocomposites obviously decreased in UV region while increased in visible region (400-700 nm) (Figure S9, Supporting Information), which


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was attributed to the shift of Fermi level in EG-TiO 2 nanocomposites, Thus, the recyclability test of this SERS biosensor was carried out by irradiation using a Xe light source (Asahi Spectra, LAXC100) through the filters (460 nm

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